Prokaryotic toxin-antitoxin systems play a major role in the maintenance of genetic information and in response to stress. The toxin of a toxin-antitoxin pair has cytostatic capabilities and usually affects cell growth or leads to cell death. The toxin and its activity can be neutralized in cells containing a plasmid that encodes for the corresponding antitoxin for the toxin. Toxin-antitoxin systems thus contribute to the maintenance of the extrachromosomal genetic information in bacterial populations by interfering selectively with the growth or viability of plasmid-free segregants or of host cells that are about to produce plasmid-free segregants if their growth is not arrested. Such systems are also found in the chromosomes of bacteria and archaea where they can have different functions, such as regulation of cell growth and viability under different stress conditions.
The Kid-Kis toxin-antitoxin system of the plasmid R1 shows significant similarities with other existing toxin-antitoxin systems (Diago-Navarro et al., FEBS Lett 277, 3097-3117 (2010)). This toxin-antitoxin pair is encoded by the parD locus of the Escherichia coli plasmid R1, which is conserved in closely related plasmids like R100. In addition, there are also chromosomal homologues in Escherichia coli, which are functionally and structurally related to other known toxins-antitoxins. For the toxin-antitoxin pair Kid and Kis it was shown that the Kid toxin could specifically inhibit cell proliferation and viability in eukaryotic cells (De la Cueva Mendez et al., EMBO J 22, 246-251 (2003)). When both genes for the toxin Kid and the antitoxin Kis are expressed, the cells grow in a normal manner. If the concentration of the Kid toxin exceeded a certain ratio to the concentration of the Kis antitoxin or if the antitoxin gene expression was repressed, growth was inhibited and the cells subsequently died by apoptosis as a result of the Kid activity. Similar results were also reported for other toxin-antitoxin pairs (Kristoffersen et al., Appl Environ Microbiol 66, 5524-5526 (2000); Yamamoto T A et al., FEBS Lett 519, 191-194 (2002)).
The observation that toxin-antitoxins can be used for selective cell killing or diminishing cell growth in prokaryotic or eukaryotic cells makes toxin-antitoxin systems suitable for treatment of degenerative disorders, such as cancer.
US 2009/0075270 A1 describes methods for evading the action of Escherichia coli Kid (PemK), and for manipulating nucleic acid expression. In particular, target sites for Kid/PemK endoribonuclease were identified and mutated.
The US 2009/0124012 A1 describes compositions and methods for regulating cell growth and metabolism by expression of components of toxin-antitoxin pairs. The described system is used for decreasing the cellular growth rate and comprises a first nucleotide sequence encoding an mRNA interferase operably linked to a first heterologous regulatory element, wherein the expression of the nucleotide sequence diminishing the growth rate of the target cells.
EP 1 198 239 B1 describes a composition comprising the parD Kid toxin and parD Kis antitoxin, for use in a therapeutic method of inhibiting cell proliferation and/or cell cycle progression carried out on a human or animal body. The method comprises providing within eukaryotic cells in the human or animal body the toxin and antitoxin, under appropriate control for selective cell cycle inhibition and/or killing of target cells.
The ParD system has also been used to investigate a role of germ line in the sex differentiation in zebra fish during somatic development. The expression of Kid toxin eliminated selectively primordial germ cells, whereas the uniform expression of the Kis antitoxin protected somatic cell lines (Slanchev K et al., Proc Natl Acad Sci USA 102, 4074-4079 (2005)).
Although the observations that toxin-antitoxin essentially could be used for diminishing the cell growth rate or cell killing in both prokaryotic and eukaryotic cells, no approach or system has been described so far that could be applied to successfully treat a pathological disease by specifically killing pathologic prokaryotic or eukaryotic cells. It would hence be desirable to selectively kill target cells, cancer cells for instance, by a toxin-antitoxin combination in order to treat pathological conditions. In order to achieve this goal, it would be necessary to specifically target cells (e.g. pathologically affected cells) with a toxin-antitoxin combination in order to increase the concentration of the toxin over the antitoxin in the target cells for killing or to increase the concentration of the antitoxin in non-target cells to protect them from the activity of the toxin. Alternatively, the expression of the antitoxin could be repressed in order to obtain an excess of the toxin in the target cell.
Shi Ya-Li et al., US National Library of Medicine (NLM), Bethesda, Md., US (May 2008), RelE toxin protein of Mycobacterium tuberulosis induces growth inhibition of lung cancer A-549 cell) teaches RelE, RelB and RelE genes subcloned into PcDNA3. The recombinant vectors were used to transfect lung cancer A-549 cells by liposome transfection. Said genes were under the control of a heterologous promoter. This expression system, however, does not allow the controlled alteration of the ratio of an toxin/antitoxin pair within target and/or non-target cells. A similar expression system for Rel proteins is also disclosed in S. B. Korch et al., Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules inhibit mycobacterial growth an dare expressed in infected human macrophages, Journal of Bacteriology, Vol. 191, No. 5, Dec. 29, 2008.
Also other publications merely address the issue of cell growth inhibition by expressing or inhibiting the a toxin and/or an antitoxin of a toxin-antitoxin pair in target cells, but do not address the actual ratio of toxin/antitoxin within the target-cells and/or non-target cells (see K. Nehlsen et al., Toxin-antitoxin based transgene expression in mammalian cells, Nucleic Acids Research, Vol. 38, No. 5, Dec. 8, 2009; F. F. Correia et al., Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli, Journal of Bacteriology, Vol. 199, No. 24, Oct. 13, 2006K; Mathieu Picardeau et al., The spirochetal chpK-chromosomal toxin and antitoxin locus induces growth inhibition of yeast and mycobacteria, FEMS Microbiology Letters, Vol. 229, No. 2, Dec. 1, 2003).
Another problem that needs to be faced is that the toxin-antitoxin members must be specifically delivered to the targeted cells in order to express their function or activity specifically in these cells. Different approaches have been employed for aiming a specific delivery of drugs, chemicals and other compounds to target cells, among these are viruses, plasmids, polymer particles or nanocells. These delivery systems are able to carry cell cycle inhibitors to the targeted cells while avoiding toxicity to non-targeted cells. Bacterially-derived minicells have been described for targeted delivery of chemotherapeutic drugs (Mac Diarmid et al., Cancer Cell, 11, 431-445 (2007)). Minicells were first observed and described by Howard Adler and colleagues in 1967 who also created the term “minicell” for bacterially-derived nanocells. Minicells are non-living nano-sized cells (approximately 200-400 nm in diameter) and are produced as a result of mutations in genes that control normal bacterial cell division, thereby de-repressing polar sides of cell division. Since the size of the vector is 200-400 nm in diameter, the term “nanocell”, as used in the present invention, is often used instead of the term “minicell”.
It was demonstrated that a range of chemotherapeutic drugs with differing structure, charge, hydrophobicity and solubility such as 5-fluoracil, carboplatin, cisplatin, doxorubicin, irinotecan, paclitaxel and vinblastine could be readily packaged within the minicells. Although the potency of minicells to deliver chemotherapeutic drugs to target cells constitutes a promising approach, specificity and cell targeting still remains as a problem. MacDiarmid et al delivered minicells to cancer cells using bispecific antibodies. The problem however was that the utilized antibodies were difficult and costly to prepare. Thus, bispecific antibodies did not provide the results that would be satisfactory for therapy or diagnosis.
It would therefore by desirable to have a nanocell-based delivery system by which not only toxin-antitoxin compounds could be transported to the respective target cells, but also any other drug, antigen, chemical, protein or whatsoever that needs to be delivered to pathological cells.
A further problem that needs to be faced is the immunogenicity of the nanocells since they are of bacterial origin. So far, a systemic administration could result in unwanted side-effects as bacterial products are known to elicit potent inflammatory responses activated by bacterial proteins and structures. A separation procedure has been developed to eliminate free endotoxin and free bacterial components to minimize the potential of toxic side-effects (MacDiarmid et al., Cancer Cell, 11, 431-445 (2007)).